This aims of this proposal are to better understand the basic mechanisms of gene expression in eukaryotes. Specifically, the mechanism of transcription by RNA Polymerase II (Pol II), the enzyme responsible for expression of all protein coding genes in eukaryotes will be dissected in the model system Saccharomyces cerevisiae. During transcription, Pol II must undergoes a continuous cycle of substrate selection and RNA synthesis directed by a DMA template. The mechanisms of Pol II substrate selection are not well understood nor are the mechanisms of Pol II regulation by elongation factors that control the activity of Pol II. This proposal will determine residues of the Pol II active site required for substrate specificity using biochemical approaches to characterize Pol II mutant enzymes. Additionally, preliminary evidence suggests that the elongation factor TFIIS, has previously unrecognized functions in modulating the elongation process. Functional interaction between a TFIIS mutant that allows its distinct functions to be separated and Pol II will be examined. These experiments will significantly advance our knowledge of the transcription process, which is the engine for gene expression in living cells. Many cellular processes from development to response to environmental signals have large or essential gene expression components, for which normal Pol II is absolutely required. As such, in many disease states Pol II function is misregulated causing aberrant gene expression that may be crucial for disease pathology. This study in particular chooses to study the highly conserved Pol II from the simple baker's yeast, S. cerevisiae. This system provides a unique platform for combining genetic, biochemical and structural approaches to understanding the mechanism of Pol II function. Insights into Pol II function will allow a better understanding of how Pol II may be misregulated in disease states in other species, such as humans. [unreadable] [unreadable] [unreadable]